CN101883568B - Nutraceutical compositions containing stevia extract or stevia extract constituents and uses thereof - Google Patents

Abstract

The invention relates to a novel nutraceutical composition containing Stevia extract or its constituents, such as steviol and stevioside, as active ingredient(s). The term ''nutraceutical'' as used herein denotes usefulness in nutritional, pharmaceutical and veterinary fields of application. The compositions are useful for improvement of cognitive functions, such as learning, memory and alertness, and psychotic stability.

Description

Novel nutraceutical composition containing stevia extract or components of stevia extract and use thereof

field of invention

The present invention relates to novel nutraceutical compositions or food additives comprising Stevia extract or components thereof such as steviol and stevioside as active ingredients for improving cognitive functions such as learning, memory and alertness, and release of psychosocial stress.

Background of the invention

Memory, learning and alertness rely on neuronal circuits in the midbrain, specifically the hippocampus, where information is processed and memories consolidated. Mental performance and learning depend on synaptic plasticity; that is, the strengthening of neuronal connections by recruiting new receptors, forming new synapses and ultimately generating new neuronal connections.

The formation of (long-term) memories and efficient functioning of the brain depend on the synthesis of new proteins that strengthen the strength of communication between neurons. The production of new proteins for synaptic strengthening is triggered by chemical and electrical signals within neurons.

Long-term potentiation (LTP) is the term used to describe long-lasting potentiation (hours in vitro, days or weeks in vivo) of synaptic transmission during short, conditional bursts of presynaptic electrical stimulation (approximately 100 Hz lasts 1 second) and then occurs at specific synapses in the central nervous system (CNS). This phenomenon is widely recognized as one of the main mechanisms by which memories are formed and stored in the brain. LTP has been observed both in vitro and in living animals. Under experimental conditions, applying a series of short, high-frequency electrical stimuli to synapses can strengthen chemical synapses for minutes to hours. Most importantly, LTP contributes to synaptic plasticity in living animals, providing the basis for a highly adaptive nervous system.

The LTP process primarily involves two distinct receptor types, the N-methyl-D-aspartate (NMDA) receptor complex and the α-amino-3-hydroxy-5-methyl-4-isoxazole Propionate (AMPA) receptors. During LTP, the major excitatory neurotransmitter glutamate is released from presynaptic neurons, binds and activates AMPA receptors on the postsynaptic membrane, causing their depolarization. At resting membrane potential, NMDA receptor channels are blocked by magnesium ions, but depolarization of the postsynaptic membrane removes this block, allowing activation of NMDA receptors and subsequent entry of calcium into the cell. This increase in intracellular calcium is believed to activate protein kinases, leading to gene transcription and enhanced protein construction (Niehoff (2005), The Language of Life: How Cells Communicate in Health and Disease, 210-223), and to enhanced AMPA receptors. Somatosensitivity, thus further promoting neurotransmission and maintenance of LTP.

The NMDA receptor is composed of NR1- and NR2-subunits; the glutamate-binding domain is formed at the junction of these subunits. In addition to glutamate, NMDA receptors also require the co-agonist glycine to regulate receptor function. The glycine binding site is present in the NR1 subunit, while the NR2 subunit has a binding site for polyamines, which are regulatory molecules that regulate NMDA receptor functionalization.

Thus, glycine is known to act as a positive allosteric modulator and obligate co-agonist together with glutamate on the NMDA receptor complex (Danysz and Parsons 1998 Pharmacol. Rev., 50(4): 597 -664). The glycine transporter (GlyT) plays an important role in the termination of postsynaptic glycinergic actions and the maintenance of low extracellular glycine concentrations by reuptake of glycine into presynaptic nerve terminals or glial cells. Termination of glycine action is thus largely mediated by rapid reuptake. Two glycine transporters, GlyT1 and GlyT2, are known and characterized by 12 putative transmembrane regions, while three GlyT1 variants (GlyT1a, b, and c) encoded by the same gene were identified (Borowsky and Hoffman 1998 J. Biol Chem., 273(44):29077-29085).

GlyT1 is the only sodium chloride-dependent glycine transporter in the forebrain that is coexpressed with NMDA receptors. At this site, GlyT1 is thought to be responsible for controlling extracellular glycine levels at synapses (López-Corcuera et al 2001 Mol.Membr.Biol., 18(1):13-20), resulting in interference with NMDA receptor function regulation.

Indeed, in the presence of the selective GlyT1 antagonist N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)]propylsarcosine (NFPS), in mice Enhanced NMDA receptor responses in CA1 pyramidal cells were observed after Schaffer collateral stimulation in rat hippocampal slices (Bergeron, et al 1998, Proc.Natl.Acad.Sci.USA, 95(26) : 15730-15734). In vivo systemic administration of NFPS in adult mice increased LTP in the dentate gyrus and enhanced prepulse suppression of the acoustic startle response, suggesting that inhibition of GlyT1 affects NMDA receptor function in a behaviorally relevant manner ( Kinney, et al 2003, J. Neurosci., 23(20):7586-7591).

Such data highlight the usefulness of compounds capable of localizing neuropathic syndromes by increasing synaptic NMDA receptors in normal individuals and for maintaining or enhancing physiological cognitive functions such as memory and learning. Extracellular glycine levels in the microenvironment to enhance synaptic function of NMDA receptors.

There is increasing interest in the development of compounds and nutraceutical compositions that can be used in the elderly and young, individuals whose daily tasks require particularly high memory and concentration (including students, construction workers, drivers, pilots, physicians, salespersons, administrators, housewives, "high-performance professionals"), and those under mental or daily stress, and those prone to nervous instability (such as schizophrenia) Improve learning, memory and alertness.

Therefore, compounds or nutraceutical compositions that enhance NMDA receptor function and enable improved learning, memory and alertness would be highly desirable.

Stevia extract has been added to beverage/food compositions in the past (see for example Japanese Kokai 2005-278467 by Nippon Paper Chemical Co., Ltd), but the stevia extract acts as a sweetener.

Detailed description of the invention

It has been discovered according to the present invention that compounds of the following formulas I and II are activators of hippocampal function through their ability to induce LTP and are useful as nutraceuticals and/or pharmaceuticals for enhancing cognitive function. These compounds may act by inhibiting glycine reuptake by inhibiting the glycine transporter GlyT1, or by activating another pathway leading to LTP induction, or by both mechanisms. Accordingly, the present invention relates to methods of enhancing cognitive function by administering to animals, including humans, a cognitive function enhancing amount of a compound of formula I, II or a mixture thereof.

According to the present invention, it is also found that stevia extract can inhibit glycine reuptake by inhibiting glycine transporter GlyT1. The resulting increase in extracellular glycine levels leads to enhanced NMDA receptor activation, the first step in inducing transcriptional activation of numerous genes and subsequently LTP, a major cellular mechanism involved in memory formation and consolidation.

Furthermore, it was found according to the present invention that the compounds steviol, isosteviol and stevioside, which can be found in stevia extracts, induce LTP through different mechanisms. Since both processes have the same biological benefit, ie they both promote hippocampal function, another aspect of the invention is the use of one or more of these active ingredients to enhance cognitive function.

Accordingly, one aspect of the present invention is a novel nutraceutical composition for enhancing cognitive function comprising stevia extract or one or more compounds of formula I or II. In addition to isosteviol, especially preferred compounds of Table 1 are steviol and stevioside.

Formula I: See Table 1 for details of the R groups.

Table 1: Structures of steviol and related glycosides (Formula I). Glc, Xyl and Rha represent glucose, xylose and rhamnose fragments, respectively (Kuznes of (2007) Steviol glycosides-chemical and technical assessment, 68 ^th JECFA meeting).

Compound name R1 R2 Steviol h h Steviolbioside h β-Glc-β-Glc(2→1) Stevioside β-Glc β-Glc-β-Glc(2→1) Rebaudioside A β-Glc β-Glc-β-Glc(2→1)|β-Glc(3→1) Rebaudioside B h β-Glc-β-Glc(2→1)|β-Glc(3→1) Rebaudioside C (dulcoside β-Glc β-Glc-α-Rha(2→1)|

(dulcoside) B) β-Glc(3→1) Rebaudioside D β-Glc-β-Glc(2→1) β-Glc-β-Glc(2→1)|β-Glc(3→1) Rebaudioside E β-Glc-β-Glc(2→1) β-Glc-β-Glc(2→1) Rebaudioside F β-Glc β-Glc-3-Xyl(2→1)|β-Glc(3→1) Rubusoside β-Glc β-Glc Durcoside A β-Glc β-Glc-α-Rha(2→1)

Formula II: Isosteviol (CAS No. 27975-19-5) is a decomposition product of steviol glycoside.

Compounds of formula I and II can be produced synthetically using known methods, can be extracted from natural sources such as plants using known extraction procedures, or they can be used as components of plant extracts, preferably as components of stevia extracts For use, the stevia extract contains sufficient steviol and/or stevioside to be an effective enhancer of hippocampal function.

Figure overview

Figure 1 shows the dose-response curve of Stevia extract in the GlyT1 inhibition assay. Assay results are expressed as % inhibition of internalization of radioactive glycine into cells. Figure 1 clearly demonstrates that Stevia extract is able to specifically inhibit the action of GlyT1 in a cellular assay.

Figure 2a shows the error rate of the results in the step-down test; a: Significantly different from vehicle-treated age-matched littermates during the training period. b: Significantly different from vehicle-treated age-matched littermates during the test period. c: Significantly different from vehicle-treated age-matched littermates during the washout cycle. d: Significantly different from Ginkgo biloba-treated mice during the washout cycle. In all cases, significance was noted at p<0.05. These data showed that the stevia-treated mice not only learned better than the other groups, but also retained their memories for a longer period of time.

Figure 2b shows the duration of the latency (shock escape) step-off behavior test. b: Significantly different from vehicle-treated age-matched littermates during the test period. c: Significantly different from vehicle-treated age-matched littermates during the washout cycle. d: Significantly different during the scavenging cycle compared to Ginkgo-treated mice. In all cases, significance was noted at p<0.05. Thus, mice treated with 150 mg/kg stevia extract showed significantly better learning and memory performance than their age-matched controls, while at the highest dose, stevia-treated mice showed significantly better learning and memory performance than age-matched control mice as well as ginkgo Both treated positive controls performed better.

Figure 3a demonstrates the latency to locate the previous location of the hidden platform in the Morris water maze during the training day. a: Significantly different from vehicle-treated age-matched littermates during the first day, emphasizing that treatment with low dose stevia extract (50 mg/kg) significantly improved the animals' learning performance. Significance is marked as p<0.05.

Figure 3b: Latency to locate the previous location of the hidden platform in the Morris water maze on test day (day 4). a: Significantly different from vehicle-treated age-matched littermates, showing that treatment with medium (150 mg/kg) and high (450 mg/kg) doses of stevia extract significantly improved memory performance in animals to the same extent as administration of the reference substance The same was observed in Ginkgo-treated mice, better than in rolipram-treated animals. Significance is marked as p<0.05.

Figure 4a shows error rates in the shuttle box test: a. Significantly different from vehicle-treated age-matched littermates during the test period. b: The period of extinction period is significantly different from vehicle-treated age-matched littermates. Significance is marked as p<0.05. Thus, at the highest dose examined during the test period, Stevia-treated animals showed significantly better memory performance than vehicle-treated littermates, and equivalent performance to mice administered the positive control compound rolipram.

Figure 4b demonstrates escape times in the shuttle box test: a: Significantly different from vehicle-treated age-matched littermates during the test period. b: Significantly different from Ginkgo-treated age-matched littermates during the test period. Significance is marked as p<0.05. These results show that stevia-treated mice (at doses of 150 and 450 mg/kg) learned to escape foot shocks better than both vehicle-treated control mice and mice administered the reference substance rolipram .

Figure 5 shows the duration of visits in each corner 3 hours before and 3 hours after the item's appearance. Arrows indicate placing items. Ginkgo and Stevia extracts significantly increased the duration of visits to corners containing novel items compared to vehicle-treated control mice (p=0.004 and p=0.005, respectively). Both treatments thus increased the exploratory activity of the mice in the object recognition test.

Figure 6 shows the location error rate (percentage of incorrect corners visited). Stevia extract treatment resulted in significantly lower percent error rates compared to both vehicle-treated control mice and Ginkgo-administered mice (p=0.012 and p=0.017, respectively). This effect was observed from the beginning of the module and was maintained for the first 12 hours (active period), demonstrating that treatment with stevia resulted in higher attention levels and improved memory performance.

Figure 7 shows the side error rate (percentage of incorrect sides with the nose touching the correct corner). Stevia extract again induced significantly lower percent error rates compared to vehicle-treated control and Ginkgo-treated mice (p=0.002 and P=0.035, respectively). This effect was observed from the beginning of the block and was maintained for the first 12 hours (active period), indicating improved attention and memory in Stevia-treated mice.

stevia extract

Stevia extracts can be produced from any species of the genus Stevia such as Stevia rebaudiana, Steviaeupatoria, Stevia ovata, Stevia plummerae, Stevia salicifolia and Steviaserrata. Typically the stevia extract should contain at least about 10-95%, preferably about 40-85%, steviosides and steviols.

As used throughout the specification and claims, the term "stevia extract" is intended to be used broadly and may include extracts of the plant produced by conventional means such as steam distillation, water-based extraction, Alcohol extraction, or extraction and ion exchange chromatography based on organic solvents such as ethyl acetate.

The only critical parameters about stevia extract are:

1) It should be acceptable for use in nutraceutical compositions intended for animal or human consumption. Solvents to be used in their preparation should therefore be approved by various regulatory agencies for the intended use. Therefore, the preferred extraction supplies are hot water, steam, alcohol and ethyl acetate.

2) It should contain sufficient amounts of steviosides, isosteviols and/or steviols to be effective.

In addition, dried plant parts (eg leaves) were found to contain sufficient amounts of the active ingredient. Therefore, stevia in dry form is also intended to be included in the present invention as a valuable source of stevia extract and components thereof. Stevia extracts typically contain other compounds that may also be biologically active and/or increase the bioavailability of the active ingredients of Stevia. Their amount present in the Stevia extract can vary based on a number of factors including: the Stevia species used, the growing conditions of the plant, and (of course) the method used to prepare the Stevia extract. A typical stevia extract prepared using an aqueous extraction method will contain steviol, isosteviol, stevioside, and rebaudioside.

Stevia extract and its components benefit mental state

As mentioned earlier, memory, learning and mental stability are based on LTP or strengthening of neuronal connections, which occurs through activation of AMPA and NMDA receptors in the brain, especially in the hippocampus. As glycine reuptake inhibitors, stevia extracts through their activity on GlyT1 allow glycine to accumulate near NMDA receptors, which in turn activates said receptors and ultimately leads to the activation of LTP (a major cellular mechanism involved in memory formation and memory consolidation). induced.

Additionally, steviol, isosteviol, and stevioside also induce activation of the same biochemical pathway (albeit at a different step than stevia extract), leading to LTP induction and are similarly beneficial for improving memory function.

Therefore, stevia extract and its components can activate hippocampal function and improve memory formation and consolidation, as well as improve mental health. Thus, as used throughout the specification and claims, the term "cognitive function enhancing amount" means a dose sufficient to activate hippocampal function and capable of resulting in improved cognitive function, as explained further below.

The improved situation of the present invention:

In the context of the present invention, "treatment" also includes co-treatment as well as prophylaxis, attenuation of symptoms associated with a particular condition, reduction of the duration of onset of a particular condition, reduction of the severity of a condition and reduction of the likelihood that an asymptomatic individual will manifest the condition in the future sex.

Throughout the specification and claims, the term "improved cognitive function" means a condition that supports and maintains cognitive health and balance, such as:

· Enhanced learning, including:

o Language processing

o solve problems

o Intellectual function

· Ability to cope with social and psychological burdens

· Enhanced attention and concentration

· Enhanced memory and memory skills, especially short-term memory

Increased mental alertness and alertness, reduction in mental fatigue

Stabilization of mental status, including:

o Alleviate postpartum conditions

оRelieve psychological burden due to separation from spouse, children, death of a loved one or marital problems

о Mitigation of issues related to residence, job change or similar events

оReducing stressful situations after traffic accidents or other negative social stress

Reduce stress, including:

o Treat, prevent and ameliorate symptoms associated with work overload, exhaustion and/or "burnout"

o Increased resistance or tolerance to pressure

o Facilitates and promotes relaxation in normal healthy individuals

· "Condition improvement" includes:

o Reduced irritability and fatigue

o Ability to cope with new situations

o Reduce, prevent or relieve physical and mental fatigue

o Promoting good quality sleep in diseased or healthy individuals, that is to say combating insomnia and sleep disorders and more generally improving energy.

In a preferred aspect of the invention, the composition may be used as a nutritional supplement, especially for persons who feel a need for enhanced cognitive function and/or psychosocial support. A non-exhaustive list of people who would benefit from enhanced cognitive function includes:

elder,

students or persons preparing for examinations,

Children who are busy with a lot of learning, i.e. infants, toddlers, preschoolers and school-going children,

Construction workers, or operators of potentially dangerous machinery,

truck drivers, pilots, train drivers or other transportation professionals,

air freight manager,

Salespeople, executives, and other "productivity professionals"

police officers and military personnel,

housewife,

or anyone exposed to a great deal of stress in their daily work, or individuals whose daily work requires exceptionally high concentration/concentration/high mental and mental performance, e.g. athletes, chess players, golfers, professional performers (actors , musicians, etc.).

To achieve these improvements, administration over several days (eg at least 6 or 10 days) is recommended, and daily administration is usually preferred for several weeks.

In addition to human applications, the compositions of the present invention have additional uses in the veterinary field. Animals that may benefit from enhanced cognitive function include those animals subjected to stressful situations. Such conditions occur, for example, after capture or transport, or may be due to housing conditions (e.g. a change of residence or owner), when the animal develops a similar condition or is distressed or aggressive, or displays stereotypic behaviour, or Anxiety and obsessive-compulsive behavior. Animals under stress should also include racing animals (eg, dogs, horses, camels), or animals used in a variety of sports, performance animals (eg, circus animals and animals appearing on stage, on television, or in movies), and performance animals Horses for equestrian and other highly disciplined daily tasks.

Preferred "animals" are pet or companion animals and farm animals. Examples of pets are dogs, cats, birds, ornamental fish, guinea pigs, (wild) rabbits, hares and ferrets. Examples of farm animals are aquaculture fish, pigs, horses, ruminants (cattle, sheep and goats) and poultry.

Nutraceutical use/formulation/dosage

As used herein, the term "nutraceutical" denotes usefulness in both the fields of nutrition and pharmacology. Thus, the novel nutraceutical composition can be used as a food and drink supplement and as a pharmaceutical formulation for enteral and parenteral application, which can be a solid formulation such as capsules or tablets, or a liquid formulation such as a solution or suspension.

The nutraceutical composition according to the invention may further contain protective hydrocolloids (e.g. gums, proteins, modified starches), binders, film formers, encapsulants/materials, wall/pore materials, matrix compounds, coatings, Emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, vehicles, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, Taste masking agents, weighting agents, jellyfying agents, gel formers, antioxidants and antimicrobial agents.

In addition, multi-vitamin and mineral supplements may be added to the nutraceutical composition of the present invention to obtain adequate amounts of essential nutrients, which are missing in some diets. Multivitamin and mineral supplements can also be used for disease prevention and to prevent lifestyle-induced nutrient losses and deficiencies.

The nutraceutical composition according to the invention may be in any galenical form suitable for administration into the body, in particular any form convenient for oral administration, for example solid forms such as fortified food or feed, food or feed premix, food or feed (additives/supplements), tablets, pills, granules, lozenges, capsules and planar formulations such as powders and tablets, or in liquid form such as solutions, emulsions or suspensions such as drinks, pastes and oily suspensions. Pastes may be incorporated into hard or soft shell capsules with a matrix such as (fish, porcine, poultry, bovine) gelatin, vegetable protein or sulfoligin. Examples of other application forms are transdermal, parenteral or injectable administration. Dietary and pharmaceutical compositions may be in the form of controlled (delayed) release formulations.

Examples of foods are dairy products including eg margarine, spreads, butter, cheese, soft milk or milk drinks.

Examples of fortified foods are sweet corn, bread, cereal bars, baked goods such as cakes and cookies, and potato chips or chips.

Beverages include non-alcoholic drinks and alcoholic drinks, and liquid preparations to be added to drinking water and liquid food. Non-alcoholic drinks are eg soft drinks, sports drinks, fruit juices, lemonades, teas and milk-based drinks. Liquid foods are eg soups and dairy products. Nutraceutical compositions containing stevia extract, its components or enriched stevia extract may be added to soft drinks, energy bars or candies such that an adult individual consumes 1 to 1000 mg, more preferably 50-750 mg, most preferably 100-500 mg of stevia extract, its fractions or enriched stevia extract.

If the nutraceutical composition is a pharmaceutical formulation, the composition also contains pharmaceutically acceptable excipients, diluents or adjuvants. Their formulation may use standard techniques, eg, Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, PA, USA. For oral administration, tablets and capsules are preferably used which contain suitable binders such as gelatin or polyvinylpyrrolidone, suitable fillers such as lactose or starch, suitable lubricants such as magnesium stearate, and optionally Optionally contain other additives. Preference is given to formulations containing 10 to 750 mg, more preferably 50 to 500 mg of stevia extract or components thereof per administration unit (eg per tablet or capsule).

For the purposes of the present invention, suitable daily dosages of stevia extract or components thereof for animals including humans may range from 0.15 mg per kg body weight to about 10 mg per kg body weight per day. More preferred is the nutraceutical composition of the present invention preferably containing Stevia extract or its components in an amount sufficient to be administered to an adult (about 70 kg in body weight) from about 10 mg/day to about 750 mg/day, preferably Doses from about 50 mg/day to about 500 mg/day.

The following non-limiting examples are provided to better illustrate the invention.

Example 1

Composition and preparation of stevia extract

A typical stevia aqueous extract disclosed in the present invention contains:

Stevioside About 70%

Rebaudiosides About 20%

Steviol, Isosteviol About 10%

Mix the ground Stevia rebaudiana leaves with hot water for 20-30 minutes. The aqueous extract is then removed by draining, using pressure to achieve maximum extraction. Several types of infusion/drainage processes can be used. Allow the extract to cool to room temperature. To remove particulates, the extract can be allowed to stand while the particulate material is allowed to settle, or the extract can be centrifuged. The extract is subsequently dried by spray drying or freeze drying. The extract contains sweetener ingredients, phytochromes, and other water-soluble components.

Stevia extract ("Stevia leaf extract, 85% powder"; catalog number S1964) used in the following examples was purchased from Spectrum Laboratory Products Inc., Gardena, CA, USA.

Example 2

Inhibition of glycine transporter 1 in a cellular assay

CHO cells stably expressing human glycine transporter 1b cDNA (GlyT1) were routinely cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, USA) containing 10% dialyzed fetal bovine serum, penicillin, streptomycin proline, and the antibiotic G418. Cells were harvested by trypsinization the day before the assay and plated in the medium described above. Immediately before the assay, the medium was replaced with an uptake buffer containing 150 mM NaCl, 1 mM CaCl ₂ , 2.5 mM KCl, 2.5 mM MgCl ₂ , 10 mM glucose, and 10 mM N-2-hydroxyethylpiperazine-N'-2 - Ethylsulfonic acid (HEPES buffer).

Glycine uptake into cells was determined by adding 60 nM radiolabeled [ ^3H ]glycine (Amersham Biosciences GE Healthcare, Slough, UK) and incubating for 30 minutes at room temperature. Incorporated glycine was quantified by liquid scintillation counting after removal of unincorporated tag by three gentle washes with the above buffer.

The addition of stevia extract inhibits glycine uptake via the GlyT1 transporter in a dose-dependent manner. Sarcosine, ORG24598 and ALX5407 (all from Sigma, St. Louis, USA) were used as known GlyT1 inhibitors. Measured _IC50 values for inhibition of glycine uptake (Stevia extract, extract fractions and reference compound) and representative dose-response curves (Stevia extract) are shown in Table 2 and Figure 1, respectively.

Table 2 : _IC50 values measured for the inhibition of glycine uptake into CHO cells by stevia extract and its main components, steviol, isosteviol and stevioside, in addition to the reference compounds sarcosine, ORG24598 and ALX5407. Data are expressed as mean ± standard deviation (where more than _one experiment was performed); where _IC50 is expressed in μM for pure compounds and in μg/ml for extracts.

substance _IC50 of tritiated glycine uptake Stevia extract (composition according to Example 1) 45±6.9μg/ml Steviol (Chromadex; catalog number ASB-00019352-025 ) Inactive

Isosteviol (Wako Chemicals; catalog number 090-02341 ) Inactive Stevioside (Indofine Chemical Co.; catalog number 023718 ) Inactive Sarcosine (Sigma; catalog number S7672) 35.9μM ORG24598 (Sigma; catalog number O7639) 0.02μM ALX5407 (Sigma; catalog number A8977) 6.46nM Rebaudioside A Inactive Ginkgo biloba (Huisong Pharmaceuticals, GB 102-070116) Inactive

Example 3

hippocampal slice culture

Seven-day-old Wistar rats were decapitated using a circular knife. In less than 1 minute the skull is opened, _the cerebral hemispheres are separated and transferred, and each hippocampus is excised and transferred into ice-cold buffer containing 137 mM NaCl, 5 mM KCl, 0.85 mM _Na2HPO4 , 1.5 mM CaCl ₂ , 0.66 mM KH ₂ PO ₄ , 0.28 mM MgSO ₄ , 1 mM MgCl ₂ , 2.7 mM NaHCO ₃ , 1 mM Kynurenic acid and 0.6% D-glucose.

Transverse hippocampal slices (400 μm) were prepared using a vibrating blade microtome (VT1200S; Leica Microsystems (Schweiz) AG, Heerbrugg, Switzerland) in the same buffer. Hippocampal slices were each placed on membrane inserts (Millicell Culture Plate Inserts, 0.4 μm) and incubated at 35° C., 5% CO ₂ , and 95% humidity in the following atmosphere containing a 1:1 mixture of BME and MEM (both from Invitrogen). Cultivate in culture medium, described medium contains the horse serum of 25% heat inactivation, 1x GlutaMAX, 1x penicillin/streptomycin, 0.6% glucose and 1mM kynurenic acid (Stoppini 1991J.Neurosci.Methods 37 (2) : 173-82).

After culturing for 48 hours, in 140 mM NaCl, 5 mM KCl, 1.3 mM CaCl ₂ , 25 mM HEPES (pH 7.3), 33 mM D-glucose and 0.02 mM bicuculline methiodide, by adding stevia extract, its The main component or control substance was used for 15 minutes to activate synaptic NMDA receptors. Sarcosine (100 μM) and ALX5407 (20 nM) were routinely used as positive controls. An additional positive control included the addition of 200 μM glycine to sister medium. After processing, sections were washed and fixed for immunohistochemical analysis. Quantification of markers of enhanced synaptic activity (see Table 3 below), which is often accompanied by long-term potentiation, represents an ex vivo model of learning and memory.

Table 3. Relative changes in synaptic markers after treatment with stevia extract or some of its component compounds (steviol, isosteviol, stevioside, and rebaudioside A) compared to sister cultures treated with buffer activation. Activation of any of these markers (or combinations thereof) is observed in a classical LTP assay.

substance pCREB pMAPK GluR1 Stevia Extract ± + 255% Steviol ++ ± 340% Isosteviol + ± 361% Stevioside ± ++ ± Rebaudioside A ± ± ± ginkgo ± + ±

++++ shows quantified maximal activation, ++ and + indicate half-maximal and moderate activation, respectively, while ± indicates no change in immunoreactivity; for GluR1, values are shown as a percentage increase of the control value.

Treatment of hippocampal cultures with stevia extract, steviol, isosteviol, and stevioside induced one or more biochemical markers typical of LTP (pCREB: activated form of cAMP response element-binding protein; pMAPK: activated form of Mitogen-activated protein kinase; GluR1: present on the cell surface of AMPA receptor 1).

Example 4

Effects of Stevia Extract in Three Traditional Rodent Models of Learning and Memory:

1- Step down test

A Combined Learning and Memory Paradigm-Stand-Off Test in Mice. The mice were placed individually in a reaction box whose floor was equipped with a 36V power grid. When animals receive an electric shock, their normal response is to jump onto an insulated platform to avoid the painful stimulus. Consequently, most animals that step down onto the net will quickly jump back onto the platform after receiving the shock. Animals were trained for 5 min on day 0, and the number of times each mouse was shocked (i.e., made a mistake) was recorded. The data constitute learning data. Mice were retested after 24 hours (day 2, training day) and 48 hours (day 2, test day), and these trials functioned as memory tests. Record the number of animals in each group that were shocked, the time until the first jump off the platform (latency), and the number of errors during the first 3 min. Six days after the end of training, memory decline was tested on day 8 (clearance test).

The study contained 6 test groups (n=12 per group). The test substances or vehicle were administered by daily oral gavage (10 ml/kg) for 30 days before training the animals. The stevia extract was tested at 3 doses of 50 (low), 150 (medium) and 450 (high) mg/kg, while the reference substance Ginkgo was administered at a dose of 100 mg/kg. The pharmacological positive control rolipram (0.1 mg/kg) was administered by intraperitoneal injection 30 minutes before the test.

All groups of animals treated with stevia showed significantly better learning and memory performance during the training and memory period and after the washout period, as indicated by a reduction in error rates, compared to vehicle-treated littermates (negative control) (Fig. 2a). In addition, during the training period, stevia-treated mice (50 mg/kg) performed as well as mice administered the control compound Ginkgo biloba, and performed better than rolipram-treated mice; during the test period, all Three doses of stevia extract resulted in similar performance to that observed in mice treated with ginkgo and rolipram; during the washout period, mice administered low and medium doses of stevia extract were also treated with the reference substance Mice treated with the positive control performed as well, while the highest dose of stevia induced significantly better performance than that observed in ginkgo-treated mice.

During the test period, latency was significantly increased in mice administered medium doses of Stevia extract compared to vehicle-treated control mice. In addition, this effect was equivalent to that observed in rolipram-treated mice. During the washout period, the highest dose of Stevia extract induced a significant increase in latency compared to vehicle-treated control mice and Ginkgo extract-administered mice (Fig. 2b).

Thus, in conclusion, stevia extract was able to improve learning and memory performance to a similar or better extent than the natural reference substance Ginkgo biloba and the pharmaceutical positive control compound rolipram.

Figure 2a: Error rate of results in the step-off test; a: Significantly different from vehicle-treated age-matched littermates during the training period. b: Significantly different from vehicle-treated age-matched littermates during the test period. c: Significantly different from vehicle-treated age-matched littermates during the washout period. d: Significantly different during the scavenging cycle compared to Ginkgo-treated mice. In all cases, significance was noted at p<0.05. These data showed that the stevia-treated mice not only learned better than the other groups, but also retained their memories for a longer period of time.

Figure 2b shows the results of the step-off behavior test; the duration of the latency to escape from the shock. b: Significantly different from vehicle-treated age-matched littermates during the test period. c: Significantly different from vehicle-treated age-matched littermates during the washout cycle. d: Significantly different during the scavenging cycle compared to Ginkgo-treated mice. In all cases, significance was noted at p<0.05. Thus, mice treated with 150 mg/kg stevia extract showed significantly better learning and memory performance than their age-matched controls, while at the highest dose, stevia-treated mice showed significantly better learning and memory performance than age-matched control mice and ginkgo biloba Treated positive controls performed better.

Example 5

Effects of Stevia Extract in Three Traditional Rodent Models of Learning and Memory:

2-Morris Water Maze

The Morris water maze is one of the best accepted paradigms for testing spatial memory in rodents. Mice must swim in a circular pool and find hidden platforms with the aid of visual cues used in the laboratory.

The same doses and treatment times as in the step-down test paradigm were used here.

The Morris water maze (diameter = 1.5m) used in this study contained a hidden platform (10cm x 10cm) located 10cm from the edge of the pool in the northwest quadrant (at 10 o'clock). On day 0, mice were habituated to the test field and received two training trials during which they were trained to swim to the platform.

On days 1 to 3, the platform was removed from the water maze and mice were subjected to two training trials per day. After each training session (90 seconds), the platform was returned to the same position and the mice were allowed to find and climb the platform. This protocol ensures that mice efficiently learn the location of the platform on each training day and recall the location of the platform on subsequent trials. Record the initial time to locate the platform placement area (first platform crossing time; FCPT) and the number of times the mouse swims through the platform placement area (number of crossings), and provide the average score for each training day (day 1 to day 3: training score). On day 4, the mice received two more trials, the results of which were the test scores.

On day 1 of the training session, mice administered the lowest dose of stevia performed significantly better than vehicle-treated control mice and mice administered the positive control compound rolipram (Fig. 3a). Mice treated with medium and high doses of stevia showed significantly shorter latency times compared to age-matched control animals during the test trial on day 4 (Fig. 3b). These data therefore indicate that treatment with stevia extract affects learning and memory performance in mice and that this effect is dose dependent.

Figure 3a shows the latency to locate the previous location of the hidden platform in the Morris water maze during the training day. a: Significantly different from vehicle-treated age-matched littermates during the first day, emphasizing that treatment with low dose stevia extract (50 mg/kg) significantly improved the animals' learning performance. Significance is marked as p<0.05.

Figure 3b shows the latency to locate the previous location of the hidden platform in the Morris water maze on the test day (day 4). a: Significantly different from vehicle-treated age-matched littermates, showing that treatment with medium (150 mg/kg) and high (450 mg/kg) doses of stevia extract significantly improved memory performance in animals to the same extent as administration of the reference substance The same was observed in Ginkgo-treated mice, better than in rolipram-treated animals. Significance is marked as p<0.05.

Example 6

Effects of Stevia Extract in Three Traditional Rodent Models of Learning and Memory:

3- Shuttle box test

The paradigm on which this animal testing is based is active avoidance of adverse stimuli. Part of the shuttle box houses the grid that delivers the shocks. The shock is preceded by an auditory signal ("beep" noise, lasting 7 seconds). Animals must learn to escape to the other side of the shuttle box, thus crossing the infrared beam.

The same doses and treatment times as in the step-down test paradigm were used here.

Mice were trained once a day for 5 days. The training program consisted of 15 intervals; each interval consisted of (1) a 15-second pause, (2) a 7-second auditory signal ("beeping period"), and (3) an 8-second electric shock ("stimulation period"). "). If the mouse crosses one of the two infrared beams at either end of the shuttle box during the alarm period or the stimulation period, proceed to the next interval. Record the number of active escapes (alarm period) and negative escape times (stimulus period), and calculate the total number of escapes. Record as an error if the mouse did not cross the infrared beam during the alarm period. The number of mistakes and escapes recorded on the 4th day was used as the training score, the 5th day was used as the test score, and the 8th day was used as the exhaustion score.

During the test period (day 5), the highest dose of stevia extract induced significantly fewer errors compared to vehicle-treated control mice; the effect on error rate was the same as in rolipram-treated positive control mice observed to be equivalent. Additionally, medium and high doses of stevia extract significantly decreased escape time compared to vehicle- and ginkgo-treated mice. Therefore, these data also show that treatment with stevia extract affects the learning performance of mice, but this effect is dependent on the dose used.

Figure 4a shows error rates in the shuttle box test: a. Significantly different from vehicle-treated age-matched littermates during the test period. b: The period of extinction period is significantly different from vehicle-treated age-matched littermates. Significance is marked as p<0.05. Thus, at the highest dose examined during the test period, Stevia-treated animals showed significantly better memory performance than vehicle-treated littermates, and equivalent performance to mice administered the positive control compound rolipram.

Figure 4b demonstrates escape times in the shuttle box test: a: Significantly different from vehicle-treated age-matched littermates during the test period. b: Significantly different from Ginkgo-treated age-matched littermates during the test period. Significance is marked as p<0.05. These results show that stevia-treated mice (at doses of 150 and 450 mg/kg) learned to escape foot shocks better than both vehicle-treated control mice and mice administered the reference substance rolipram.

Example 7

Effects of Stevia Extract in a New, Fully Automated Rodent Model of Learning and Memory

中比较用甜菊提取物或银杏处理的小鼠的认知表现，所述系统允许在居住笼样环境(homecage-like environment)中自动监测小鼠的自发行为和学习行为(NewBehavior AG，Zürich，Switzerland，www.newbehavior.com；Galsworthy et al.2005，Behav Brain Res 157：211-217；Onishchenko et al.2007，Toxicol Sci 97：428-437)。个体小鼠被IntelliCage角落中读取应答器(参照识别标签)的传感器识别，所述应答器植入小鼠的颈背中。每个

实际上是一个大的笼子(37.5x 55x20.5cm)，向其中放置包含四个记录(工作)室的金属框架。记录室安置在笼的角落，各自覆盖地板空间的15x 15x 21cm直角三角型区域。笼内天线允许自动化监测每只个体小鼠的角落访问；每个角落内的光束允许自动化记录个体的鼻子触碰(nosepoke)和舔水瓶口(licks of the water bottlespout)行为。将四个三角型小鼠掩蔽体置于笼中心，在上面放置食物漏斗，使得能够随意取用食物。exist

Comparing the cognitive performance of mice treated with stevia extract or ginkgo biloba in The system allows automatic monitoring of spontaneous and learned behavior in mice in a homecage-like environment (NewBehavior AG, Zürich, Switzerland, www.newbehavior.com; Galsworthy et al. 2005, Behav Brain Res 157: 211 -217; Onishchenko et al. 2007, Toxicol Sci 97:428-437). Individual mice are identified by sensors in the corners of the IntelliCage that read transponders (referenced to identification tags) that are implanted in the scruff of the mouse's neck. each

Actually a large cage (37.5x 55x20.5cm) into which was placed a metal frame containing four recording (working) chambers. Recording chambers were placed at the corners of the cage, each covering a 15x 15x 21 cm right-angled triangular area of floor space. Antennas within the cage allow automated monitoring of corner visits for each individual mouse; light beams within each corner allow automated recording of individual nosepoke and licks of the water bottlespout behavior. Four triangular mouse shelters were placed in the center of the cage, on which a food funnel was placed to allow ad libitum access to food.

Each recording chamber contained: (1) a plastic ring (30 mm inner diameter) that served as an entrance into the chamber and covered the loop antennas accessed by the recording corners; (2) a gridded floor on which the mice sat once they entered the chamber above; (3) two annular openings (13 mm diameter) which enable access to the mouth of the water bottle; each opening is passed through by a light beam registering a nose touch; (4) two motorized doors which allow (door open) or (5) two water bottles; (6) tubing through which a puff of air can be delivered as an unpleasant stimulus to the mouse; (7) LEDs of different colors which can be used to for adjustment experiments.

Experimental stage :

The study contained three test groups (n=12-14 each): vehicle (control), ginkgo (100 mg/kg) and stevia extract (450 mg/kg). All mice were administered test substance or vehicle by daily oral gavage (10 ml/kg) throughout the study period of 8 weeks.

During the habituation period (4-5 days), mice had free access to all corners, water and feed, and were free to explore the cage. Subsequently, the mice had to learn to apply the nose touch (nose touch adaptation module, 3 days); all doors were initially closed (no access to water), and the mice had to perform the nose touch to open the doors and reach the mouth of the water bottle. The collected data contains several parameters, such as the least preferred corner of each individual mouse, which are recorded for programming the next module.

item identification

To test intrinsic exploratory activity in mice, two identical objects were placed in corners 1 and 2 or in corners 3 and 4, respectively, including the least preferred corner of each group. Animals had free opportunity to explore the cage and had full access to water and feed. Visits were recorded 3 hours before and 3 hours after the item was present. Access patterns in the control group were unchanged following the presentation of the item in corners 1 and 2, whereas both Ginkgo and Stevia treatments resulted in significantly increased access duration to the corners containing novel items (corners 4 and 2, respectively) (Figure 5). Thus, chronic administration of stevia extract significantly increased exploratory activity in mice to the same extent as Ginkgo biloba.

Figure 5 shows the duration of visits in each corner 3 hours before and 3 hours after the item's appearance. Arrows indicate placing items. Ginkgo and Stevia extracts significantly increased the duration of visits to corners containing novel items compared to vehicle-treated control mice (p=0.004 and p=0.005, respectively). Both treatments thus increased the exploratory activity of the mice in the object recognition test.

Side Discrimination

This module is designed to test attention and associative memory. Assign a correct corner to each mouse. Only specifying one side (out of two) in this corner is correct and is indicated to the animal by a green LED. Animals were able to nose touch on the correct side and then drink from the water bottle. Place errors (ie percentage of incorrect corners visited) and side errors (ie percentage of nose touches on incorrect sides of correct corners) were recorded during this module.

These data indicate improved attention following chronic treatment with stevia extract as place error rates (Figure 6) and side errors compared to vehicle and Ginkgo treated mice during the first 12 hours of testing (active period). rate (Figure 7) was significantly lower.

Figure 6 shows the location error rate (percentage of incorrect corners visited). Stevia extract treatment resulted in significantly lower percent error rates compared to both vehicle-treated control mice and Ginkgo-administered mice (p=0.012 and p=0.017, respectively). This effect was observed from the beginning of the module and was maintained for the first 12 hours (active period), demonstrating that treatment with stevia resulted in higher attention levels and improved memory performance.

Figure 7 shows the side error rate (percentage of nose touching the incorrect side of the correct corner). Stevia extract again induced significantly lower percent error rates compared to vehicle-treated control and Ginkgo-treated mice (p=0.002 and P=0.035, respectively). This effect was observed from the beginning of the block and was maintained for the first 12 hours (active period), indicating improved attention and memory in Stevia-treated mice.

All in all, from automated The results of the study validated the behavioral results from the classic tests (Examples 4, 5 and 6): 8 weeks of treatment with stevia extract resulted in a significant improvement in learning and memory in mice compared to vehicle-treated littermates. In some instances, the performance improvements observed in Stevia-treated mice were even greater than those observed in mice administered either the natural reference substance Ginkgo biloba or the pharmaceutical positive control compound rolipram.

Example 8

Preparation of Soft Gelatin Capsules

Prepare soft gelatin capsules containing the following ingredients:

Element Amount Per Capsule Stevia Extract (freeze-dried/spray-dried) 200mg Lecithin 50mg Soybean oil 250mg

Adults may be administered two capsules per day for 3 months. Improvements in cognitive function, alertness, and the ability to focus on work were observed.

Example 9

Prepare ready-to-eat flavored soft drinks

Element Quantity [g] Stevia Extract 0.5 Sucrose, fine powder 763.1 Ascorbic acid, fine powder 2.0 Citric acid, anhydrous powder 55.0 Lemon Spice 8.0 Trisodium citrate, anhydrous powder 6.0 Tricalcium phosphate 5.0 β-Carotene 1% CWS from DNP AG, Kaiseraugst, Switzerland 0.4 Total 840

All ingredients were mixed and sieved through a 500 μm sieve. The resulting powder was placed in a suitable container and mixed in a tubular mixer for at least 20 minutes. To prepare a drink, 105 g of the mixed powder obtained are mixed with enough water to prepare one liter of drink.

The ready-to-drink soft drink contains about 15mg enriched stevia extract per serving (250ml). As a strengthener or for general health, take 2 servings (500ml) per day. Improvements in cognitive function, alertness, and the ability to focus on work were observed.

Example 10

Preparing Fortified No-Bake Cereal Bars

Element Quantity [g] Stevia Extract 0.3 water 54.0 Salt 1.5 glucose syrup 130.0 invert syrup 95.0 Sorbitol Syrup 35.0 palm kernel oil 60.0 Baking Fat 40.0 Lecithin 1.7 hardened palm oil 2.5 Dried and sliced apples 63.0 Corn Crisp 100.0 Rice crispies 120.0 Wheat crispies 90.0 Roasted Hazelnuts 40.0 Skimmed milk powder 45.0 Apple Flavor 74863-33 2.0 citric acid 5.0 Total 885

Rosemary extract was premixed with skim milk powder and placed in a planetary bowl mixer. Add corn crisps and rice crisps and mix the whole gently. Then add dried and cut apples. In the first cooking pot, mix water and salt in the amounts given above (solution 1). In a second cooking pot, mix the glucose syrup, invert syrup and sorbitol syrup in the amounts given above (solution 2). The fat phase consists of a mixture of baking fat, palm kernel fat, lecithin and emulsifiers. Solution 1 was heated to 110°C. Solution 2 was heated to 113°C and then cooled in a cold water bath. Solution 1 and Solution 2 were then combined. Melt the lipid phase in a water bath at 75°C and add to the combined mixture of solutions 1 and 2. Add the apple flavor and citric acid to the liquid sugar/fat mixture. Add liquid ingredients to dry ingredients and mix well in planetary bowl mixer. Place material on marble slab and roll to desired thickness. Cool the mass to room temperature and cut into pieces. Unbaked cereal bars contain about 10mg of stevia extract per serving (30g). For general health and energy, consume 1-2 cereal bars per day. Improvements in cognitive function, alertness, and the ability to focus on work were observed.

Example 11

Dry Dog Food with Stevia Extract

50)或L-抗坏血酸钠/钙的三磷酸酯、二磷酸酯和单磷酸酯的混合物(例如来自DSM Nutritional Products Ltd，Kaiseraugst，瑞士的

35)一起，对狗用商业基础膳食(例如Mera Dog“Brocken”，MERA-Tiernahrung GmbH，Marienstraβe 80-84，D-47625Kevelaer-Wetten，德国)进行喷雾。将食物组合物干燥至含有按重量计约90％的干物质。对于每天消耗约200g干饲料的10kg体重的平均狗而言，狗食物含有每kg食物约200mg甜菊提取物。对更重的狗而言，相应地制备饲料混合物。为了在狗中减少压力、恐惧和攻击性，可以在动物掩蔽所(animal shelter farm)中有规律地给予所述食物。在看兽医之前或在兽医诊所停留或假期分离时，在应激事件之前至少一周、事件期间和之后一周给予所述食物。With the amount that is enough to administer the daily dose of 4mg stevia extract per kg body weight of dog, with the solution of stevia extract in water and antioxidant such as vitamin C (for example from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland C-EC) and its derivatives, i.e. sodium ascorbyl monophosphate (e.g. from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland

50) or a mixture of triphosphate, diphosphate and monophosphate of sodium/calcium L-ascorbate (for example from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland

35) Together, a commercial basal diet for dogs (eg Mera Dog "Brocken", MERA-Tiernahrung GmbH, Marienstra βe 80-84, D-47625 Kevelaer-Wetten, Germany) is sprayed. The food composition is dried to contain about 90% dry matter by weight. For an average dog of 10 kg body weight consuming about 200 g of dry feed per day, dog food contains about 200 mg of stevia extract per kg of food. For heavier dogs, prepare the feed mix accordingly. To reduce stress, fear and aggression in dogs, the food can be given regularly in an animal shelter farm. The food is given at least one week before, during and one week after a stressful event prior to a veterinary visit or while staying at a veterinary clinic or on holiday separation.

Example 12

Moist cat food with stevia extract

C-EC)及其衍生物，即抗坏血酸单磷酸钠(例如来自DSM Nutritional Products Ltd，Kaiseraugst，瑞士的

50)或L-抗坏血酸钠/钙的三磷酸酯、二磷酸酯和单磷酸酯的混合物(例如来自DSM Nutritional Products Ltd，Kaiseraugst，瑞士的

35)一起，与猫用商业基础膳食(例如Happy Cat“Adult”，Tierfeinnahrung，Südliche Hauptstraβe 38，D-86517Wehringen，德国)混合。对于消耗约400g湿润食物的5kg体重的平均猫而言，猫食物含有每kg食物50mg甜菊提取物。将食物组合物干燥至含有按重量计约90％的干物质。为了在猫中减少压力、恐惧和攻击性，可以在动物掩蔽所中有规律地给予所述食物。在看兽医之前或在兽医诊所停留时，在应激事件之前至少一周、事件期间和之后一周给予所述食物。In an amount sufficient to administer a daily dose of 4 mg stevia extract per kg body weight to cats, a solution of stevia extract in water with an antioxidant such as vitamin C (such as from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland

C-EC) and its derivatives, i.e. sodium ascorbyl monophosphate (e.g. from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland

50) or a mixture of triphosphate, diphosphate and monophosphate of sodium/calcium L-ascorbate (for example from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland

35) Together, mixed with a commercial base diet for cats (eg Happy Cat "Adult", Tierfeinnahrung, Südliche Hauptstraβe 38, D-86517 Wehringen, Germany). For an average cat of 5 kg body weight consuming about 400 g of moist food, the cat food contains 50 mg of stevia extract per kg of food. The food composition is dried to contain about 90% dry matter by weight. To reduce stress, fear and aggression in cats, the food can be given regularly in animal shelters. The food is given at least one week before, during, and one week after a stressful event prior to a veterinary visit or a visit to a veterinary clinic.

Example 13

Dog Treats with Stevia Extract

C-EC)及其衍生物，即抗坏血酸单磷酸钠(例如来自DSM Nutritional Products Ltd，Kaiseraugst，瑞士的

50)或L-抗坏血酸钠/钙的三磷酸酯、二磷酸酯和单磷酸酯的混合物(例如来自DSM Nutritional Products Ltd，Kaiseraugst，瑞士的35)一起，对商业狗零食(例如由MeraTiernahrung GmbH，Marienstrasse 80-84，47625Kevelaer-Wetten，德国提供的狗用Mera Dog“Biscuit”)进行喷雾。将食物组合物干燥至含有按重量计约90％的干物质。为了减少恐惧和紧张，可以在一天中除食物之外给予零食，或者在不保证饲喂时(即旅行期间)每天至多5次给予零食。In an amount sufficient to administer 0.5-5 mg of stevia extract per g of snacks to the dispenser, a solution of stevia extract in water and an antioxidant such as vitamin C (for example from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland)

C-EC) and its derivatives, i.e. sodium ascorbyl monophosphate (e.g. from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland

50) or a mixture of triphosphate, diphosphate and monophosphate of sodium/calcium L-ascorbate (for example from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland 35) Together, commercial dog treats (eg Mera Dog "Biscuit" for dogs supplied by MeraTiernahrung GmbH, Marienstrasse 80-84, 47625 Kevelaer-Wetten, Germany) are sprayed. The food composition is dried to contain about 90% dry matter by weight. To reduce fear and nervousness, treats can be given throughout the day in addition to food, or up to 5 times a day when feeding is not warranted (i.e. during travel).

Example 14

Cat Treats with Stevia Extract

C-EC)及其衍生物，即抗坏血酸单磷酸钠(例如来自DSM Nutritional Products Ltd，Kaiseraugst，瑞士的

50)或L-抗坏血酸钠/钙的三磷酸酯、二磷酸酯和单磷酸酯的混合物(例如来自DSM Nutritional Products Ltd，Kaiseraugst，瑞士的

35)一起，对商业猫零食(例如由Whiskas，Masterfoods GmbH，Eitzer Str.215，27283Verden/Aller，德国提供的猫用Whiskas Dentabits)进行喷雾。将食物组合物干燥至含有按重量计约90％的干物质。为了减少恐惧和紧张，可以在一天中除食物之外给予零食，或者在不保证饲喂时(即旅行期间)每天至多5次给予零食。In an amount sufficient to administer 0.5-5 mg of stevia extract per g of snacks to the dispenser, a solution of stevia extract in water and an antioxidant such as vitamin C (for example from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland)

C-EC) and its derivatives, i.e. sodium ascorbyl monophosphate (e.g. from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland

50) or a mixture of triphosphate, diphosphate and monophosphate of sodium/calcium L-ascorbate (for example from DSM Nutritional Products Ltd, Kaiseraugst, Switzerland

35) Together, commercial cat treats (such as Whiskas Dentabits for Cats supplied by Whiskas, Masterfoods GmbH, Eitzer Str. 215, 27283 Verden/Aller, Germany) are sprayed. The food composition is dried to contain about 90% dry matter by weight. To reduce fear and nervousness, treats can be given throughout the day in addition to food, or up to 5 times a day when feeding is not warranted (i.e. during travel).

Claims (3)

1. in manufacture, for the purposes in the nutrient drug/medicine that improves study, memory and vigilance the animal or human, described extract contains the Flos Chrysanthemi aqueous extract of cognitive function improvement amount:

About 70% stevioside,

About 20% Rebaudiodside A,

Approximately 10% steviol and isosteviol, and

Plant pigment and other water-soluble components.

2. according to the purposes of the extract of claim 1, wherein said nutrient drug or medicine are applied 6-10 days.

3. in manufacture, for the purposes in the product that improves study, memory and vigilance the animal or human, described extract contains the Flos Chrysanthemi aqueous extract:

About 70% stevioside,

About 20% Rebaudiodside A,

Approximately 10% steviol and isosteviol, and

Plant pigment and other water-soluble components.

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